Fungal endophytes
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Fungal endophytes

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of infection (n \ufffd 5
leaves). We then assessed pathogen damage by determining leaf
mortality (proportion of leaves that were abscised or consumed
entirely by necrosis) and the area of damage on surviving leaves
(proportion of leaf area showing necrosis, measured using a leaf-
area meter). A paired t test was used to compare rates of mortality
for E\ufffdP\ufffd and E\ufffdP\ufffd leaves. Due to differential leaf mortality and
treatment allocation, unpaired analyses were used to compare
damage on surviving leaves.
Endophyte Abundance and Richness in T. cacao. Naturally infected
leaves of T. cacao were characterized by numerous and dense
endophyte infections. Endophytes were present in sampled
tissue from 82.2%, 95.6%, and 100% of young, mature, and old
leaves, respectively (n \ufffd 135 leaves). Density of endophyte
infections in mature and old leaves approached 100% of leaf
segments infected and exceeded that in young leaves by a factor
of two. In contrast, mature leaves of seedlings grown under
protected conditions contained endophytic fungi in\ufffd1% of leaf
segments (Fig. 1). Consistent with previous studies (24, 25), these
data provide strong evidence that endophytes associated with T.
cacao are transmitted horizontally.
From 126 naturally infected leaves of T. cacao, we recovered
1,172 endophyte isolates representing 344 morphotaxa. The most
commonly isolated morphotaxa (n \ufffd 20) comprised 60% of iso-
lates; all other morphotaxa were rarely encountered. Leaf samples
comprising 32 mm2 per leaf (representing \ufffd5% of total leaf area;
ref. 25) contained up to 13 distinct taxa of endophytic fungi. Fungal
taxa used in subsequent seedling inoculations (Colletotrichum sp.,
Fusarium\ufffdNectria spp., and Xylaria sp.) co-occurred frequently
in asymptomatic tissues. Colletotrichum sp. M1 was the most
frequently encountered endophyte, occurring in 71.9 \ufffd 6.8% of
mature and old leaves per tree (range: 33\u2013100%).
Species richness of endophytes increased significantly with
leaf age, ranging from 4.48\ufffd 0.46 to 6.23\ufffd 0.45 and 8.69\ufffd 0.31
morphotaxa per leaf for young, mature, and old leaves, respec-
tively (\ufffd2 \ufffd 37.35, P \ufffd 0.0001). Richness of endophytes recov-
ered from individual trees (n \ufffd 9 leaves per tree) ranged from
38.3\ufffd 3.8 to 47.5\ufffd 4.9 morphotaxa per tree. Despite abiotic and
biotic differences among sites (see below), richness of endo-
phytes associated with focal trees did not differ significantly with
regard to site (F4,10 \ufffd 2.34, P \ufffd 0.1256).
Spatial Structure of Endophytic Fungi. Similarity in endophyte
assemblages decreased as a curvilinear function of distance
between survey sites (R2\ufffd 0.993, F3, 6\ufffd 287.06, P\ufffd 0.0001; Fig.
2). Highest similarities occurred between T. cacao in intact forest
(BCI) and in an abandoned plot under secondary forest at PNS
(the closest site to BCI; MH\ufffd 0.928, JI\ufffd 0.458). These sites also
share the most complex overstory and are similar in terms of
rainfall regime. Relatively high values also were found for
comparisons between BCI and a recently abandoned plot of T.
cacao cultivated under scattered shade trees at ND, the second-
closest site to BCI (MH\ufffd 0.714, JI\ufffd 0.387). These sites are\ufffd60
km from one another and differ in plant diversity, species
composition, annual rainfall, and duration of the dry season. In
contrast, comparisons between ND and an active plantation
(BT), which are separated by \ufffd325 km but are similar in terms
of general land use, rainfall, and plant diversity, approached zero
(MH \ufffd 0.023, JI \ufffd 0.023).
Host Affinity of Endophytes. Concurrent surveys of T. cacao, H.
concinna, and O. lucens at BCI indicated that endophytes were
present in 100% of mature leaves and leaf segments of all focal
hosts. Among endophyte taxa recovered from more than one
leaf, 65.5% were encountered in only one host species. Ten
endophyte taxa were found concurrently in two or three host
species; however, their relative abundances differed with regard
to host. For example, Colletotrichum sp. M1 occurred in 88.9%
of cacao leaves, but was recovered from only 33.3% of leaves of
O. lucens, and was never recovered from H. concinna. When
endophyte assemblages were compared on the basis of species
composition and isolation frequencies (MH), endophytes asso-
ciated with conspecific hosts within individual sites, or among
sites separated by 20\u201360 km, were more similar to one another
than were endophyte assemblages associated with three host
species at one site (F2,24 \ufffd 3.85, P \ufffd 0.0356; Fig. 3). These data
Fig. 1. Fungal endophytes associated with leaves of T. cacao are horizontally
transmitted and accumulate over leaf lifetimes. Cultivable endophytes were
not found in surface-sterilized seeds (data from ref. 24) and occurred in\ufffd1%
of tissue segments of mature leaves of seedlings (100 days old) raised under
sterile conditions. Under field conditions, proportions of leaf segments (each
2 mm2) containing endophytes increased with leaf age (mean\ufffd SE; data from
BT, ND, PNS, and BCI; n \ufffd 3 leaves per age class per site).
Fig. 2. Endophytic fungi associated with T. cacao demonstrate spatial
structure. Similarity of endophyte assemblages, defined by the abundance-
based MH for endophyte taxa occurring in more than one leaf, decreased as
a function of distance between hosts. Points represent MH obtained in 10
pairwise comparisons of endophyte assemblages associated with T. cacao in
each of five sites (n\ufffd 9 leaves per tree, 3 trees per site; P\ufffd 0.0001; JI data are
congruent and are not shown).
Arnold et al. PNAS \ufffd December 23, 2003 \ufffd vol. 100 \ufffd no. 26 \ufffd 15651
are consistent with previous findings of nonrandom associations
of endophytes with H. concinna and O. lucens over a small
sampling area (33, 38), as well as data from other guilds of fungi
(42, 43).
In in vitro trials, 86% of cacao endophytes differed signifi-
cantly in growth rate when cultivated on media containing
extracts from leaves of T. cacao vs. H. concinna or O. lucens (F2,
70\ufffd 15.71, P\ufffd 0.0001). In a second experiment, growth rates for
77% of tested morphotaxa were greatest when endophytes were
cultivated on media containing extracts of the host species from
which they were most frequently isolated in field surveys (sign
test, P\ufffd 0.0348). For those taxa, growth on extracts of their most
frequent host exceeded growth on extracts of the host in which
they occurred most rarely by \ufffd20% (\ufffd2 \ufffd 13.23, P \ufffd 0.0013).
Efficacy of Inoculation. Both E\ufffd and E\ufffd leaves were successfully
produced on individual seedlings of T. cacao. Six endophyte taxa
originally isolated from asymptomatic T. cacao were successfully
reisolated from E\ufffd leaves, whereas one endophyte species
originally isolated from H. concinna (Xylaria sp.) was never
recovered from E\ufffd tissues.
After inoculation, endophyte infection densities in E\ufffd leaves
rapidly approached values comparable to those observed in field
surveys. Fourteen days after inoculation (i.e., 4 days before
infection with Phytophthora), inoculum endophytes were recov-
ered from 85.7% of E\ufffd leaves, and 37.5 \ufffd 0.1% of E\ufffd leaf
segments in infected leaves (n \ufffd 7 leaves). In contrast, only one
of 192 leaf segments assessed for E\ufffd leaves (0.5%) contained an
inoculum endophyte (n \ufffd 6 leaves). Twenty nine days after
inoculation (i.e., 10 days after infection with Phytophthora),
endophytes were recovered from all E\ufffd leaves (n \ufffd 9 leaves).
At that time, endophytes were present in 72.4\ufffd 0.1% of E\ufffd leaf
segments, indicating proliferation of inoculum taxa in leaf tissues
(percent of leaf segments infected at 14 vs. 29 days, F1, 14\ufffd 7.07,
P \ufffd 0.0187). The most common morphotaxon in our field
surveys of T. cacao (Colletotrichum sp. M1) also was the most
prevalent endophyte in E\ufffd tissues, occurring in 100% of E\ufffd
leaves sampled 29 days after inoculation. Nonetheless, 89% of
E\ufffd leaves contained two to four endophyte species, indicating
simultaneous infection by multiple taxa